Gel Filtration Chromatography- Definition, Principle, Types, Parts, Steps, Uses
Gel filtration chromatography, also known as size exclusion chromatography or molecular sieve chromatography, is a technique that separates molecules based on their size and shape. It is widely used in the purification and analysis of biomolecules, such as proteins, nucleic acids, polysaccharides and other macromolecules.
Gel filtration chromatography is a type of size exclusion chromatography, which separates molecules based on their size and shape. The principle of gel filtration chromatography is that larger molecules pass through a porous matrix faster than smaller molecules, because they have less access to the pores of the matrix.
Group separations are used to separate the components of a sample into two major groups according to their size range. For example, a group separation can be used to remove high or low molecular weight contaminants (such as phenol red from culture fluids) or to desalt and exchange buffers. Group separations are usually performed with a low-resolution medium that has a wide fractionation range (the range of molecular weights that can be separated by the medium). The advantage of group separations is that they are fast and simple, and can be used as a preparatory step for further purification or analysis.
High-resolution fractionation of biomolecules
High-resolution fractionation of biomolecules is used to separate the components of a sample according to differences in their molecular size. For example, high-resolution fractionation can be used to isolate one or more components, to separate monomers from aggregates, to determine the molecular weight or to perform a molecular weight distribution analysis. High-resolution fractionation is usually performed with a medium that has a narrow fractionation range and a high degree of selectivity (the ability to discriminate between molecules of similar size). The advantage of high-resolution fractionation is that it can achieve a high degree of separation and resolution between peaks, and can provide information about the molecular properties of the sample.
- Preparation of the column: The column is packed with the gel filtration medium of choice, which consists of spherical particles with pores of a specific size range. The column is then equilibrated with the buffer that will be used as the mobile phase. The buffer should be compatible with the sample and the gel filtration medium, and should maintain the stability and activity of the biomolecules to be separated.
- Application of the sample: The sample is applied to the top of the column using a syringe, a pump, or a sample loop. The sample volume should be small enough to avoid overloading the column and reducing the resolution. The sample should also be free of particulate matter that could clog the column or interfere with the detection.
- Elution of the sample: The buffer is continuously pumped through the column at a constant flow rate. As the buffer and the sample move through the column, they interact with the gel filtration medium. The molecules in the sample are separated based on their size and shape, as they diffuse in and out of the pores of the medium. Larger molecules are excluded from the pores and elute faster, while smaller molecules enter the pores and elute slower. The elution time of each molecule depends on its molecular weight, shape, and degree of interaction with the medium.
- Detection and collection of fractions: The eluate from the column is monitored by a detector, such as UV absorbance, fluorescence, or refractive index. The detector records a chromatogram, which shows the peaks corresponding to different molecules in the sample. The fractions containing the molecules of interest can be collected manually or automatically using a fraction collector. The fractions can then be analyzed further by other methods, such as SDS-PAGE, mass spectrometry, or enzyme assays.
Gel filtration chromatography is a versatile technique that can be used for various purposes in the field of biochemistry and molecular biology. Some of the common applications are:
- Purification of biomolecules
- Refolding of denatured proteins
- Protein fractionation
- Determination of quaternary structure
- Separation of other molecules
- Gentle and non-destructive
- Flexible and adaptable
- Simple and convenient
- Fast and efficient
- Versatile and multifunctional
- Sample volume
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